The role of ClC‐3 in volume‐activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition

Abstract

1 A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2 ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (I_Cl,vol) with an IC₅₀ of 5.3 and 9.6 mM, respectively. 3 The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4 ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5 The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6 Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7 We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8 The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between I_ClC-3 and I_Cl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells.